Disconnected optic axons persist in the visual pathway during regeneration of the retino-tectal projection in the frog

Summary In this study, we crushed one optic nerve in the frog Litoria (Hyla) moorei and at intervals thereafter anterogradely labelled optic axons with horseradish peroxidase (HRP). For one series, HRP was applied between the eye and the crush site and in a second series between the crush site and the chiasm. A tectal projection of regenerating axons was seen in both series but, in addition, up to 12 weeks post-crush, the second series displayed an additional projection. Its appearance matched that of the disconnected, but persisting, optic axon terminals which are found after enucleation or optic nerve ligation. We conclude that, in the frog, many disconnected optic axons persist throughout the period of optic nerve regeneration and of restoration of an orderly retino-tectal map.Abbreviation HRP horseradish peroxidase     

Changes in NADPH diaphorase expression in the fish visual system during optic nerve regeneration and retinal development

The various functions of nitric oxide (NO) in the nervous system are not fully understood, including its role in neuronal regeneration. The goldfish can regenerate its optic nerve after transection, making it a useful model for studying central nervous regeneration in response to injury. Therefore, we have studied the pattern of NO expression in the retina and optic tectum after optic nerve transection, using NADPH diaphorase histochemistry. NO synthesis was transiently up-regulated in the ganglion cell bodies, peaking during the period when retinal axons reach the tectum, between 20–45 days after optic nerve transection. Enzyme activity in the tectum was transiently down-regulated and then returned to control levels at 60 days after optic nerve transection, during synaptic refinement. To compare NO expression in the developing and regenerating retina, we have looked at NO expression in the developing zebrafish retina. In the developing zebrafish retina the pattern of staining roughly followed the pattern of development with the inner plexiform layer and horizontal cells having the strongest pattern of staining. These results suggest that NO may be involved in the survival of ganglion cells in the regenerating retina, and that it plays a different role in the developing retina. In the tectum, NO may be involved in synaptic refinement.     

Chronic toxic demyelination in the central nervous system leads to axonal damage despite remyelination

The majority of multiple sclerosis lesions fail to remyelinate after chronic demyelinating episodes resulting in neurologic disability. In the current study, chronic demyelination was investigated by using the cuprizone model, a toxic demyelination model. C57BL/6 mice were administered a 0.2% cuprizone diet up to 16 weeks to induce chronic demyelination. For comparison, another group was maintained only for 6 weeks on cuprizone to model acute demyelination. Both groups were analysed regarding the remyelination process after withdrawal of the toxin. Reexpression of myelin proteins after chronic demyelination was reduced by a factor of two as judged by LFB and myelin protein stainings compared to acute demyelination after 2 weeks on remyelination. During chronic demyelination mature oligodendrocytes (Nogo-A positive cells) were severely depleted by 90% compared to age matched controls. Nevertheless, extensive remyelination occurred after withdrawal of cuprizone and was nearly complete after 12 weeks. There was only minimal acute axonal damage as judged by APP staining, with the course of APP positive axons correlating with macrophage/microglia accumulation. Chronic axonal damage detected by SMI-32 positive staining was only seen after chronic demyelination and was still observable during the whole remyelination period. These data suggest that two pattern of axonal injury occur in the cuprizone model.     

Demyelination and remyelination in anatomically distinct regions of the corpus callosum following cuprizone intoxication

Multiple sclerosis is a chronic demyelinating disease of the central nervous system. Spontaneous remyelination during early disease stages is thought to preserve and partially restore function. However, this process ceases in later stages despite the presence of pre-oligodendrocytes. Cuprizone-induced demyelination is a useful model with which to study the remyelination process. Previous studies have demonstrated heterogeneities in demyelination in individual animals. Here we investigated regional differences in demyelination and remyelination within the corpus callosum. C57BL/6 mice were fed 0.2% cuprizone for 5 weeks to induce demyelination. Remyelination was examined 2-5 weeks after cuprizone withdrawal. Immunohistochemistry and electron microscopy were used to quantify regional differences in demyelination, gliosis, and remyelination. We found that, while demyelination was limited in the rostral region of corpus callosum, nearly complete demyelination occurred in the caudal callosum, beginning at approximately −0.5 mm from bregma. Astrogliosis and microgliosis were correlated with demyelination and differed between the rostral and caudal callosal structures. Remyelination upon cessation of cuprizone ensued at different rates with splenium remyelinating faster than dorsal hippocampal commissure. Our data show anatomical differences of cuprizone-induced demyelination and remyelination in the corpus callosum and the importance of examining specific callosal regions in myelin repair studies using this model.     

Topographic refinement of the regenerating retinotectal projection of the goldfish in standard laboratory conditions: a quantitative WGA-HRP study

Summary The topographic precision of the regenerating retinotectal projection of the goldfish was studied between 18 and 524 days (at 20° C) after optic nerve cut, using retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA-HRP) from one of two standardized tectal injection sites. All labelled ganglion cells in each flat-mounted retina were plotted individually, and their degree of dispersion was assessed by a statistical method based on distance to nearest neighbour. Labelled cells in normal fish were clustered tightly, covering on average only 1.3% of the retina. Early in regeneration (18–28 days) they were widely dispersed, covering up to 75.2%, and they did not begin to form recognizable clusters at appropriate sites until about 35 days after nerve cut. Between 18 and 70 days, the proportion of retina covered by labelled cells fell dramatically, halving about every 14 days. Between 70 and 524 days, no further reduction could be demonstrated: overall, clusters remained significantly larger than normal, though a few individual retinae were virtually normal. Several others, labelled from similar single injections between 56 and 524 days after nerve cut, showed pairs of cell clusters; a sign that persistent errors in topography are common. The very wide initial scatter of labelled cells reflects a striking lack of goal-directedness in regenerative axon growth. Extensive branching in the optic nerve, tract and tectum, for which there is already evidence, must contribute to this. Though uptake of some WGA-HRP by non-synaptic growth cones cannot be ruled out, other evidence for mislocated functional synapses at early stages encourages us to favour trial and error synapse formation as the likely basis of map refinement.     

Myelination of regenerated optic fibers in peripheral nerve graft of adult cats

Retinal ganglion cells of adult cats have the potential to regenerate their axons into autografted peripheral nerve. Two months after transplantation of the sciatic nerve to the axotomized optic stump, regenerated axons were labeled anterogradely with biocytin, and myelin formation by Schwann cells was examined electron microscopically. Both myelinated and unmyelinated fibers were labeled with biocytin. Among 511 axons labeled in three grafts, 96 fibers (18.8%) were myelinated and 415 (81.2%) were unmyelinated. Mean diameter with SD of myelinated fibers was 1.28 ± 0.39 m (range 0.71–2.47) and that of unmyelinated fibers was 0.76± 0.38 m (range 0.18–2.46). The ratio of inner to outer diameters of the myelin sheath (g value) was 0.82, which is close to the value (0.8) for the optic fibers of intact adult cats.     

Effect of AF64A on the cholinergic systems of the retina and optic tectum of goldfish

Summary AF64A, a presumed selective cholinergic neurotoxin has been used to study the effect on cholinergic systems of the goldfish retina and optic tectum. Toxin injection in the vitreum and in the optic tectum caused a selective decrease of choline acetyltransferase activity in both areas, while no significant decrease of glutamate decarboxylase and D-3H aspartate uptake were observed at different times after the injections. The effect was particularly dramatic in the retina of long term-injected animals, where choline acetyltransferase dropped to practically zero level. The ultrastructural analysis showed selective degeneration of some neurons in the amacrine and ganglion cell layer of the retina as well as of synaptic terminals and neuronal cell bodies in the optic tectum. The results favour a selective cholinotoxicity of AF64A in fish nerve tissue at doses substantially higher than those found to have additional unselective effects in mammals.Supported by a grant from the Italian Ministry of Education     

霍乱毒素及联合外周神经对视神经损伤后视网膜节细胞再生的影响

目的探讨霍乱毒素(CTx)及其联合外周神经对成年金黄地鼠视神经损伤后再生视网膜节细胞胞体及轴突的影响。方法扎断(MC)成年金黄地鼠视神经(ON)近端,玻璃体内注射CTx,或联合插入小段坐骨神经分支(SN),或切断视神经近端(ONT)并缝接一段自体坐骨神经,并在玻璃体内注射CTx。动物随机分为MC+CTx组、MC+CTx+SN组、ONT+SN+CTx组。各组动物均存活4周。用荧光金逆行标记再生的轴突,在荧光镜下观察视网膜平铺片中再生的视网膜节细胞大小及视神经切片内再生的轴突。结果MC+CTx组、MC+CTx+SN组、ONT+SN+CTx组再生RGCs周长依次为(56.84±18.08)μm、(83.20±28.28)μm、(94.01±32.44)μm,各组间差异有显著性。再生的RGCs有1-2个轴突,在视神经内多呈波浪状,且多走行在视神经边缘。结论各实验组促进再生的视网膜节细胞大小不同,提示霍乱毒素及其联合外周神经可能促进视网膜不同亚型节细胞再生。     

Oxidized galectin-1 advances the functional recovery after peripheral nerve injury

Oxidized galectin-1 has been shown to promote axonal regeneration from transected-nerve sites in an in vitro dorsal root ganglion (DRG) explant model as well as in in vivo peripheral nerve axotomy models. The present study provides evidence that oxidized galectin-1 advances the restoration of nerve function after peripheral nerve injury. The sciatic nerve of adult rats was transected and the distal nerve was frozen after being sutured into a proximal site with four epineurial stitches. An osmotic pump delivered oxidized galectin-1 peripherally to the surgical site. Functional recovery was assessed by measurement of the degree of toe spread of the hind paw for 3 months after the sciatic nerve lesion. The recovery curves of toe spread in the test group showed a statistically significant improvement of functional recovery after day 21 by the application of oxidized recombinant human galectin-1 (rhGAL-1/Ox) compared to the control group. This functional recovery was supported by histological analysis performed by light microscopic examination. The regenerating myelinated fibers at the site 21 mm distal to the nerve-transected site were quantitatively examined at 100 days after the operation. The frequency distribution of myelinated fiber diameters showed that exogenous rhGAL-1/Ox increased the number and diameter of regenerating myelinated fibers; the number of medium-sized (6–11 μm in diameter) fibers increased significantly (P < 0.05). These results indicate that oxidized galectin-1 promotes the restoration of nerve function after peripheral nerve injury. Thus, rhGAL-1/Ox may be a factor for functional restoration of injured peripheral nerves.     

Involvement of the histaminergic system on appetitive learning and its interaction with haloperidol in goldfish

This study investigated the actions of the histaminergic system on appetitive learning and memory, and its interaction with the dopaminergic system in goldfish. It consisted of nine sessions, in which fish were tested in a four-arm tank. On day 1, the animals were habituated for 10 min. On day 2, they were placed in one arm and had to find food at the left or the right arm. Time to begin feeding was recorded, and the procedure repeated for more 3 days (training phase). On training day 4, seven groups were injected with saline, seven with haloperidol (2.0 mg/kg) and one with DMSO solution before training and after feeding, three groups received saline, six chlorpheniramine (CPA) (1.0, 4.0 and 8.0 mg/kg), and six l-histidine (LH) (25, 50 and 100 mg/kg). Saline groups were considered as control of CPA and LH treated groups and DMSO as control of haloperidol. A non-injected group was also included. Testing occurred after 24 h. A reversal procedure was conducted 24h after testing and repeated for 3 days. The groups receiving CPA at 1.0 and 8.0 mg/kg and LH at 25, 50 and 100 mg/kg differed between Test and Reversal day 1. Pre-treatment with haloperidol plus 8.0 mg/kg of CPA and 25 and 50 mg/kg of LH reverted the treatment effect. However, in the groups treated with 1.0 mg/kg of CPA and 100 mg/kg of LH, the difference remained. This study confirmed the interaction between the histaminergic and the dopaminergic systems on memory process in goldfish.     

Monensin induces distortion of optic nerve crossing at the chick embryo chiasm

Summary The effects of intraocular injection of monensin, a specific inhibitor of membrane glycoprotein transport within the axon, on the development of the chick embryo optic nerve were examined. A proportion of axons growing from the monensin-injected eye were misrouted into the opposite optic nerve at the chiasm and grew to the retina of the opposite eye. These results suggest that formation of the decussation pattern at the chiasm primarily depends on neurite fasciculation mediated by membrane glycoproteins.     

Peripheral nerve regeneration through alginate gel: analysis of early outgrowth and late increase in diameter of regenerating axons

Our previous study revealed that alginate gel cross-linked with covalent bonds promoted peripheral nerve regeneration in the cat and rat. The present study analyzed nerve regeneration through alginate gel in the early stages within 2 weeks and the late stages up to 21 months after implantation. Four days after surgery, regenerating axons grew without Schwann cell investment through the partially degraded alginate gel, being in direct contact with the alginate without a basal lamina covering. Numerous mast cells infiltrated into the alginate. One to 2 weeks after surgery, regenerating axons were surrounded by common Schwann cells to form small bundles, with some axons at the periphery being partly in direct contact with alginate. At the distal stump, numerous Schwann cells had migrated into the alginate 8-14 days after surgery. They had no basal laminae. The diameter of regenerated myelinated fibers was small (approximately 1 micro m) at 8 weeks, but increased in diameter, having a distribution pattern similar to that of normal nerve 21 months after surgery. Much better nerve regeneration was found in alginate gel-, than collagen sponge-, and fibrin glue-implanted distal stump 12 months after surgery. These results indicate that alginate gel has good biocompatibility for regenerating axon outgrowth and Schwann cell migration, and that regenerated fibers can have a diameter as thick as that of normal fibers in the long term. Alginate gel is a promising material for use as an implant for peripheral nerve regeneration.     

Visual-vestibular interactions in the vestibular nuclei of the goldfish

Summary The responses of vestibular nuclei neurons of relaxed unaesthetized goldfish have been examined with trapezoid velocity stimuli under three conditions. Responses to horizontal body rotation in the dark (pure vestibular stimulation) resemble those observed in vestibular nerve afferents. Optokinetic responses to exclusive visual surround-motion are also direction-specific and, in contrast to vestibular responses, exhibit a tonic response to constant velocity. They show three different response profiles, classified A, B or C, based on the neuron's discharge rate: either increasing, decreasing or remaining constant once surround motion is maintained at constant velocity. Following these dynamic effects, optokinetic responses have a maintained modulation of resting discharge until deceleration commences. The time constants associated with the dynamic effects vary between 1 and 11 seconds. Steady-state modulation of optokinetic responses shows a weak relation to stimulus velocities exceeding 10 deg/sec. Responses to body rotation in the light were found to linearly combine the weighted vestibular and optokinetic responses so that accurate velocity information is available for sensory and motor functions independent of the neuron's vestibular (I, II) or optokinetic (A, B, C) response type. The principle of this visual-vestibular interaction is discussed with respect to multisensory processing within the vestibular nuclei.     

Regulatory expression of Neurensin-1 in the spinal motor neurons after mouse sciatic nerve injury

Axonal regeneration after crush injury of the sciatic nerve has been intensely studied for the elucidation of molecular and cellular mechanisms. Neurite extension factor1 (Nrsn1) is a unique membranous protein that has a microtubule-binding domain and is specifically expressed in neurons. Our studies have shown that Nrsn1 is localized particularly in actively extending neurites, thus playing a role in membrane transport to the growing distal ends of extending neurites. To elucidate the possible role of Nrsn1 during peripheral axonal regeneration, we examined the expression of Nrsn1 mRNA by in situ hybridization and Nrsn1 localization by immunocytochemistry, using a mouse model. The results revealed that during the early phase of axonal regeneration of motor nerves, Nrsn1 mRNA is upregulated in the injured motor neuron. Nrsn1 is localized in the cell bodies of motor neurons and at the growing distal ends of regenerating axons. These results indicate that Nrsn1 plays an active role in axonal regeneration as well as in embryonic development.     

GAP-43 in the axons of mammalian CNS neurons regenerating into peripheral nerve grafts

Summary Although mature mammalian CNS neurons do not normally regenerate axons after injury, it is well established that they will regrow axons over long distances into peripheral nerve implants. We have autografted segments of sciatic nerve into the brains of adult albino rats and have used light and electron microscopic immunocytochemistry to examine the distribution of the growth associated protein GAP-43 in and around the graft in the first two weeks following implantation. GAP-43 was present, 3–14 days after grafting, in small non-myelinated axonal sprouts in the brain parenchyma around the proximal tip of the graft. At 11–14 days after implantation similar sprouts within the graft itself were GAP-43 immunoreactive. The sprouts were either naked or associated with other cell processes (chiefly of Schwann cells; to a lesser extent of astrocytes). We also show that small numbers of neuronal perikarya around the tip of the graft become GAP-43 immunoreactive 11–14 days after implantation. Thus mature mammalian CNS neurons regenerating axons into a PNS graft display a marked increase in their content of GAP-43. In addition, we report that small plaques of GAP-43 reaction product are sometimes present on the plasma membranes of Schwann cells or astrocytes adjacent to immunoreactive axons, and that narrow sheet-like or filopodial processes of astrocytes, Schwann cells and possibly other non-neuronal cell types, may contain small amounts of GAP-43.     

Unconditional stimulus intensity and cardiac conditioning in the goldfish (Carassius auratus)

Independent groups of small restrained goldfish were given pairings of an illumination increase CS and shock USs of different intensities. The relation between conditioned cardio-deceleration and US intensity was a simple monotonic one with an apparent asymptote between 1.3 and 2.2 V/cm. There was no sign of cardio-acceleration at any point in adaptation, conditioning, or extinction. Baseline heart rate was little affected by conditioning but post US rate decreases mirrored the conditioned decreases.     

Study of neurotrophic activity of thrombin on the model of regenerating mouse nerve

Experiments demonstrated a dose-dependent facilitating effect of thrombin and peptide thrombin receptor agonist PAR1 (TRAP6) on regeneration of mouse peripheral nerve after its crushing. The maximum neurotrophic effect was observed at low concentrations of thrombin (10 nM) and TRAP6 (10 µM).__________This revised version was published online in August 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 8–10, January, 2005     

Glioblast migration in the optic stalk of the chick embryo

Summary Light and electron microscopy was used to study the chick embryo optic stalk from Hamburger and Hamilton stages 21 to 29. Observations of glioblast morphology in different developmental stages suggest that these cells undergo radial migration toward peripheral regions of the stalk. Immediately previous to and during migration, morphological changes were noted in the glioblasts, including the appearance of lateral prolongations which contribute to the subdivision of optic fiber fascicles and the radial elongation of their nucleus, which gives the impression of squeezing itself into the peripheral glioblastic prolongation. These phenomena occur in a retino-diencephalic direction, commencing in the distal optic stalk during stage 23 and continuing in subsequent stages. The significance of glioblastic migration is discussed in relation to possible mechanisms through which optic fiber fascicles, initially located on the surface of the stalk, come to lie in deeper areas of the stalk wall.